RESUMO
Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immunizing horses with ScNtx can successfully neutralize the lethality of pure recombinant and native short-chain α-neurotoxins, as well as whole neurotoxic elapid venoms from diverse genera such as Micrurus, Dendroaspis, Naja, Walterinnesia, Ophiophagus and Hydrophis. These results provide a proof-of-principle for using recombinant proteins with rationally designed consensus sequences as universal immunogens for developing next-generation antivenoms with higher effectiveness and broader neutralizing capacity.
Assuntos
Anticorpos/imunologia , Venenos Elapídicos/imunologia , Elapidae/imunologia , Neurotoxinas/imunologia , Sequência de Aminoácidos , Animais , Venenos Elapídicos/genética , Elapidae/genética , Cavalos , Imunização , Masculino , Camundongos , Neurotoxinas/química , Neurotoxinas/genética , Alinhamento de SequênciaRESUMO
BACKGROUND: Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. METHODS AND FINDINGS: In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. CONCLUSION: Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.
Assuntos
Antivenenos/imunologia , Antivenenos/farmacologia , Elapidae/imunologia , Venenos de Serpentes/imunologia , Tecnologia Farmacêutica/métodos , Vacinas de DNA/imunologia , Animais , Modelos Animais de Doenças , Elapidae/genética , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Feminino , Camundongos Endogâmicos BALB C , Testes de Neutralização , Mordeduras de Serpentes/terapia , Venenos de Serpentes/genética , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Venoms of snakes belonging to the same Genera tend to share biochemical, toxinological and antigenic characteristics. Accordingly, paraspecific neutralization of venom lethality by experimental antisera and commercial antivenoms has been reported. We studied the spectrum of neutralization of lethality of an experimental monovalent equine antiserum against the strongly neurotoxic African forest cobra (Naja melanoleuca) when tested against venoms of most species of African Naja, both neuro and cytotoxic as described by some authors. We report a comparison of the median lethal doses (LD50) of the venoms and the paraspecific median effective doses (ED50) of the antiserum calculated using three methods: Spearman-Kärber and Probit (currently recommended by the World Health Organization), and non-linear regression. An ample--but not complete--spectrum of paraspecific neutralization of lethality was observed against both spitting and non-spitting species of African Naja with a clearly more efficient neutralization of the more potent venoms, the implications of which are discussed. The median lethal and effective doses calculated by the three methods are remarkably consistent and may warrant consideration of non-linear regression methods for the calculation of venom lethality and antivenom potency by venom/antivenom researchers and producers.
Assuntos
Antivenenos/imunologia , Venenos Elapídicos/imunologia , Elapidae/imunologia , Soros Imunes/imunologia , Animais , Cavalos , Dose Letal Mediana , Camundongos , Testes de Neutralização , Análise de RegressãoRESUMO
Snake venom metalloproteinases (SVMPs) are present in large quantities in venoms of viper snakes and also in some elapids. Jararhagin is a representative of a P-III multidomain hemorrhagic SVMP present in Bothrops jararaca venom. It is comprised of a catalytic, a disintegrin-like and a cysteine-rich domain. Seven anti-jararhagin monoclonal antibodies (MAJar 1-7) were produced, of which six reacted with the disintegrin domain. MAJar 3 recognized an epitope present at the C-terminal part of the disintegrin-like domain, and neutralized jararhagin-induced hemorrhage. In this study, we evaluated the reactivity of these monoclonal antibodies with venoms from 27 species of snakes belonging to different families. MAJar 3 recognized most of the hemorrhagic venoms. By ELISA, MAJar 3 reacted strongly with venoms from Viperidae family and weakly with Colubridae and Elapidae venoms. This recognition pattern was due to bands between 50 and 80 kDa, corresponding to P-III SVMPs. This antibody preferentially neutralized the hemorrhage induced by venoms of Bothrops snakes. This fact suggests that the epitope recognized by MAJar 3 is present in other metalloproteinases throughout snake phylogeny. However, slight structural differences in the epitope may result in insufficient affinity for neutralization of biological activities.
Assuntos
Anticorpos Monoclonais/imunologia , Bothrops/classificação , Venenos de Crotalídeos/imunologia , Epitopos/imunologia , Hemorragia/imunologia , Metaloproteases/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting , Bothrops/genética , Bothrops/imunologia , Colubridae/genética , Colubridae/imunologia , Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/toxicidade , Elapidae/genética , Elapidae/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/genética , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Metaloproteases/química , Metaloproteases/genética , Camundongos , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Viperidae/genética , Viperidae/imunologia , Veneno de Bothrops jararacaRESUMO
Cartilha destinada às equipes de saúde e a todos que se interessam em trabalhar no controle de acidentes ofídicos